Interlaboratory diagnostic validation of conformation-sensitive capillary electrophoresis for mutation scanning
Interlaboratory diagnostic validation of conformation-sensitive capillary electrophoresis for mutation scanning
Background: indirect alternatives to sequencing as a method for mutation scanning are of interest to diagnostic laboratories because they have the potential for considerable savings in both time and costs. Ideally, such methods should be simple, rapid, and highly sensitive, and they should be validated formally to a very high standard. Currently, most reported methods lack one or more of these characteristics. We describe the optimization and validation of conformation-sensitive capillary electrophoresis (CSCE) for diagnostic mutation scanning.
Methods: we initially optimized the performance of CSCE with a systematic panel of plasmid-based controls. We then compared manual analysis by visual inspection with automated analysis by BioNumerics software (Applied Maths) in a blinded interlaboratory validation with 402 BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 1, early onset) variants previously characterized by Sanger sequencing.
Results: with automated analysis, we demonstrated a sensitivity of >99% (95% CI), which is indistinguishable from the sensitivity for conventional sequencing by capillary electrophoresis. The 95% CI for specificity was 90%–93%; thus, CSCE greatly reduces the number of fragments that need to be sequenced to fully characterize variants. By manual analysis, the 95% CIs for sensitivity and specificity were 98.3%–99.4% and 93.1%–95.5%, respectively.
Conclusions: CSCE is amenable to a high degree of automation, and analyses can be multiplexed to increase both capacity and throughput. We conclude that once it is optimized, CSCE combined with analysis with BioNumerics software is a highly sensitive and cost-effective mutation-scanning technique suitable for routine genetic diagnostic analysis of heterozygous nucleotide substitutions, small insertions, and deletions
593-602
Mattocks, Christopher
2d943111-cfdf-4f0d-9ecc-0737e541fe36
Watkins, Gemma
2de575b0-b349-40b1-a3e1-2559e416d086
Ward, Daniel
8343934a-ac4c-4042-ad30-93270144f77d
Janssens, Tom
6240c53a-3bf5-4669-9f09-bf2b886c76e2
Bosgoed, Ermanno A.J.
2c8cedc0-13c7-49a8-b14e-dd287024c150
van der Donk, Kim
7a58b9ae-c8e5-43f6-9c60-7d029860dae2
Ligtenberg, Marjolijn J.
db05b97c-a9c0-4736-9f5b-8dbde1ab2c2d
Pot, Bruno
df2d72ec-4764-404e-830b-ca583de827c8
Theelen, Joop
a4332e83-0e57-49f4-9243-a6d877e66855
Cross, Nicholas C.P.
f87650da-b908-4a34-b31b-d62c5f186fe4
Scheffer, Hans
f5bad206-92a1-4f7d-94b9-3dd8579a393d
Matthijs, Gert
c85e9151-905f-42cf-a978-66ae262202b5
April 2010
Mattocks, Christopher
2d943111-cfdf-4f0d-9ecc-0737e541fe36
Watkins, Gemma
2de575b0-b349-40b1-a3e1-2559e416d086
Ward, Daniel
8343934a-ac4c-4042-ad30-93270144f77d
Janssens, Tom
6240c53a-3bf5-4669-9f09-bf2b886c76e2
Bosgoed, Ermanno A.J.
2c8cedc0-13c7-49a8-b14e-dd287024c150
van der Donk, Kim
7a58b9ae-c8e5-43f6-9c60-7d029860dae2
Ligtenberg, Marjolijn J.
db05b97c-a9c0-4736-9f5b-8dbde1ab2c2d
Pot, Bruno
df2d72ec-4764-404e-830b-ca583de827c8
Theelen, Joop
a4332e83-0e57-49f4-9243-a6d877e66855
Cross, Nicholas C.P.
f87650da-b908-4a34-b31b-d62c5f186fe4
Scheffer, Hans
f5bad206-92a1-4f7d-94b9-3dd8579a393d
Matthijs, Gert
c85e9151-905f-42cf-a978-66ae262202b5
Mattocks, Christopher, Watkins, Gemma, Ward, Daniel, Janssens, Tom, Bosgoed, Ermanno A.J., van der Donk, Kim, Ligtenberg, Marjolijn J., Pot, Bruno, Theelen, Joop, Cross, Nicholas C.P., Scheffer, Hans and Matthijs, Gert
(2010)
Interlaboratory diagnostic validation of conformation-sensitive capillary electrophoresis for mutation scanning.
Clinical Chemistry, 56 (4), .
(doi:10.1373/clinchem.2009.135426).
(PMID:20167696)
Abstract
Background: indirect alternatives to sequencing as a method for mutation scanning are of interest to diagnostic laboratories because they have the potential for considerable savings in both time and costs. Ideally, such methods should be simple, rapid, and highly sensitive, and they should be validated formally to a very high standard. Currently, most reported methods lack one or more of these characteristics. We describe the optimization and validation of conformation-sensitive capillary electrophoresis (CSCE) for diagnostic mutation scanning.
Methods: we initially optimized the performance of CSCE with a systematic panel of plasmid-based controls. We then compared manual analysis by visual inspection with automated analysis by BioNumerics software (Applied Maths) in a blinded interlaboratory validation with 402 BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 1, early onset) variants previously characterized by Sanger sequencing.
Results: with automated analysis, we demonstrated a sensitivity of >99% (95% CI), which is indistinguishable from the sensitivity for conventional sequencing by capillary electrophoresis. The 95% CI for specificity was 90%–93%; thus, CSCE greatly reduces the number of fragments that need to be sequenced to fully characterize variants. By manual analysis, the 95% CIs for sensitivity and specificity were 98.3%–99.4% and 93.1%–95.5%, respectively.
Conclusions: CSCE is amenable to a high degree of automation, and analyses can be multiplexed to increase both capacity and throughput. We conclude that once it is optimized, CSCE combined with analysis with BioNumerics software is a highly sensitive and cost-effective mutation-scanning technique suitable for routine genetic diagnostic analysis of heterozygous nucleotide substitutions, small insertions, and deletions
This record has no associated files available for download.
More information
e-pub ahead of print date: 18 February 2010
Published date: April 2010
Identifiers
Local EPrints ID: 72745
URI: http://eprints.soton.ac.uk/id/eprint/72745
PURE UUID: 10dc5f5f-9807-4f9b-b819-acfb279f3cac
Catalogue record
Date deposited: 24 Feb 2010
Last modified: 14 Mar 2024 02:46
Export record
Altmetrics
Contributors
Author:
Christopher Mattocks
Author:
Gemma Watkins
Author:
Daniel Ward
Author:
Tom Janssens
Author:
Ermanno A.J. Bosgoed
Author:
Kim van der Donk
Author:
Marjolijn J. Ligtenberg
Author:
Bruno Pot
Author:
Joop Theelen
Author:
Hans Scheffer
Author:
Gert Matthijs
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics